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The NIH rDNA Guidelines Explained

The next few pages were written to extract the essence of the NIH guideline requirements and put them into readable form. Since many details are omitted, the actual guidelines should be consulted when in doubt (http://www4.od.nih.gov/oba/rac/guidelines.html). Plant, whole animals, and human experiments are given special treatment in the guidelines (and in this explanation).

PREREQUISITES To make sense of the following, the reader should know the basic ideas behind Biosafety Levels 1 through 4. A summary table giving the characteristics of the four laboratory Biosafety Levels is on page 6. Animal Biosafety levels are summarized on page 7.

WHAT IS RECOMBINANT DNA (rDNA)? It is either a) DNA constructed in vitro from separate DNA segments that can replicate and/or express a biologically active polynucleotide or polypeptide in vivo, or b) synthetic DNA that has the potential of generating a hazardous product in vivo.

WHAT ARE THE RAC AND THE OBA? The NIH OBA (Office of Biotechnology Activities) is an administrative arm responsible for carrying out the orders of the NIH Director with regard to recombinant DNA, genetic testing, and xenotransplantation. An advisory committee is involved in establishing policies for each of these fields. For recombinant DNA the committee is called the Recombinant DNA Advisory Committee or the “RAC”.

REGISTRATION The NIH requires all labs working with recombinant DNA to register with their local Institutional Biosafety Committee (IBC). The guidelines distinguish among the five kinds of registrations. The type depends on the potential hazard of the work. More hazardous means more approvals are needed. The five types are:

 Work that cannot begin until there is NIH and IBC approval (NIH Guidelines Sections III-A and III-B), this is the most dangerous level and it is [TABOO] (page 2)  Work that cannot begin until there is IBC and RAC review (Sections III-C). [WAIT and WAIT] (page 2) This tends to be sensitive and potentially dangerous. Human Gene Transfer studies are in this category (page5).  Work that cannot begin until there is IBC approval (III-D), there is usually a short [WAIT] (page 2).  Work can begin when the IBC is notified (Section III-E) [NO WAITING] (page 4)  Work that is Exempt form NIH Guidelines (Section III-F). No approval is needed but the work should be registered with the IBC [NO WAITING, EXEMPT] (page 4)

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“TABOO”

(NIH approval and IBC Approval Required) (from NIH Guidelines Sections III-A and III-B)

 Making drug resistant constructs of microorganisms if they compromise the drug’s therapeutic potential  Making constructs that synthesize vertebrate toxins with an LD50 of 100ng/Kg or less.  Making constructs in E. coli that synthesize vertebrate toxins that are “lethal” between 100ng/Kg and 100µg/Kg

“WAIT AND WAIT” (NIH Review and IBC Approval Required) (from Section IIIC)

Studies in which genes are transferred into humans must be submitted to the NIH OBA for review. If the OBA finds the study to be “novel” it will place the study on the next RAC meeting agenda. IBC approval must wait for the RAC’s review. If, on the other hand, OBA does not deem the study to be “novel”, IBC can act immediately.

“WAIT” (IBC Approval Needed Before Starting)

These studies are examined by the IBC with an eye to recommending safe procedures and containment. To reach a conclusion it is often useful to classify the risk associated with a proposed study according to the risk associated with the organism(s) to be used. A convenient classification tool is the concept of “risk group”.

Risk Groups The NIH classifies biological agents into four Risk Groups according to their human pathogenecity (see NIH Guidelines, Section II-A-1)

· Risk Group 1 – not associated with disease in healthy adults · Risk Group 2 – associated with disease that is rarely serious and for which therapeutic or preventive options are often available · Risk Group 3 - associated with serious or lethal disease for which therapeutic or preventive options may be available · Risk Group 4 – associated with serious or lethal disease for which therapeutic or preventive options not usually available

Appendix B in the NIH Guidelines lists a number of biologic agents according to their Risk Group. Clemson University IBC Page 3 of 8

In general, the Risk Group determines the Biosafety Level needed: for instance a Risk Group 3 agent is usually studied in a BL3 or BL3-N (animal) or BL3-P (plants) lab.

Those agents not listed in Risk Group (RGs) 2, 3 and 4 are not automatically or implicitly I RG1: a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

The table on the next page summarizes the NIH recommendations for Biosafety Level according to risk group.

“NO WAITING” Just Notify IBC Before Work Starts

Notification is necessary because all recombinant DNA studies must be registered with Clemson University.

These studies involve “Low Hazard” recombinant DNA (Section III-E)

· Non-pathogenic prokaryotes or non-pathogenic lower eukaryotes (use BL1) · Recombinant DNA with less than 2/3 of a eukaryotic viral genome (and no helper) used exclusively in tissue culture (BL1 recommended) · Some host Plants carrying recombinant DNA. BL1-P level containment is recommended when the host is: a non-noxious weed, a plant or microorganism thought not to damage the ecosystem, · Some other host Plants carrying recombinant DNA. BL3-P or BL1-P+ level containment is recommended when: the host is a noxious weed, the introduced DNA is the complete genome of an infectious agent known to normally exist in the U.S. the host is a plant or microorganism that may damage ecosystems, the host is a plant with recombinant DNA from foreign microorganisms that is thought to be safe for the ecosystem.

No Waiting - Exempt Exempt Recombinant DNA (Section III-F and Appendix C)

“Exempt” form NIH guidelines means that work with these constructs need not be approved by the IBC. However, Clemson University requires that all recombinant work, exempt or non- exempt, be registered. Thus, it is Clemson University policy to insist that investigators using any recombinant DNA register with the IBC. The exempt work can be performed at BL1.

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Gene transfer into Human subjects (RAC review process1 is required before IBC can approve this kind of study). (Section III-C-1) Recombinant organisms cultured in volumes greater than 10 liters (Section III-D-6). Pathogen Hosts (Section III-D-1): Check Appendix B to determine their Risk Group - then: Risk Group 2 host...... BL2, BL2-N Risk Group 3 host...... BL3, BL3-N Risk Group 4 host...... BL4, BL4-N Pathogen DNA Source into Non-Pathogen Host (Section III-D-2): Check Appendix B to determine their Risk Group - then: Risk Group 2 or 3 source...... BL2 Risk Group 4 source...... BL2, IF the pathogen genome is defective Risk Group 4 source...... BL4, Otherwise Animal Virus DNA source into Tissue Culture (Section III-D-3): Check Appendix B to determine their Risk Group- then: Risk Group 2 virus source...... BL2 Risk Group 3 virus source...... BL3 Risk Group 4 virus source...... BL4 Transgenic Animal2 Host (Section III-D-4): Anything but > 2/3 eukaryotic virus genome...... BL1-N (but IBC can boost this level based on the pathogenicity of the source organism) Viral vectors that don't transmit...... BL1-N Everything else is a special case...... IBC decides Modified microorganisms into Animals: (Section III-D-4): Any viable rDNA modified organism ≥BL2, BL2-N rDNA into Animals Any rDNA that (except pieces >⅔ eukaryotic viral genome) ...... BL1, BL1-N Everything else...... IBC decides Whole Plants: (Section III-D-5) Exotic3 pathogen hosts that can dam- age the ecosystem...... BL3-P or BL2-P+ Plants with transmissible DNA from exotic pathogens that can damage the ecosystem...... BL3-P or BL2-P+ Transmissible exotic pathogen hosts BL4-P Toxin DNA into plants...... BL3-P Insect Pathogen DNA that can dam- age the ecosystem...... BL3-P or BL2-P+ Genes coding for vertebrate Toxins (Appendix F)

LD50 < 100 ng/kg ...... Requires NIH & IBC approval 100 ng/kg < LD50 < 100 μg/kg ...... Requires IBC approval & NIH notifi- cation. (except in E. coli-see below) 100 ng/kg < LD50 < 1 μg/kg ...... BL2 if in E. coli 1 μg/kg < LD50 < 100 μg/kg...... BL1 if in E. coli

______1RAC can either take no action and transmit the protocol to the FDA or call for a full public review at one of its quarterly meetings. 2The purchase or transfer of transgenic rodents is exempt from the NIH guidelines. 3"Exotic" plant pathogens are defined as those not known to occur naturally in the US. ______

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Some Exempt Classes are:

· DNA vaccines encoding epitopes from microbiological sources are generally exempt from the NIH Guidelines, even in human studies. This unusual exemption is found in Appendix VI-A. · Recombinant DNA outside of living or viral organisms, · Recombinant DNA that cannot replicate or express in vivo, · DNA from a single nonchromosomal or viral source, ·The DNA source organism and the host organism are the same organism (but not released into the environment) · The DNA source organism and the host organism normally exchange DNA (organisms that normally exchange are listed in Appendix A) · DNA that does “not present a significant risk to health or the environment...” ·The recombinant DNA is used exclusively in tissue culture and has <1/2 eukaryotic viral genome. There are exceptions to this rule (Appendix C-I- A). Check with the Biosafety Officer. · Experiments using an E. coli host vector system in which the host does not contain conjugation proficient plasmids. There are some restrictions on the vectors used (Appendix C-II). BL1 containment is suggested. ·Experiments with Saccharomyces host-vector systems. There are some restrictions (Appendix C-III). BL1 containment is suggested. · Experiments with Bacillus subtilis or B. icheniformis host-vector systems and in which reversion to spore formation is <10-7. There are some other restrictions (Appendix C-IV). BL1 containment is suggested.

“THREE SPECIALIZED GUIDELINE SECTIONS”

The NIH Guidelines recognize three non-laboratory classes of Biosafety containment and procedures; those in which genes are transferred into humans (Appendix M); those for plants (BL1-P through BL4-P, Appendix P) and those for animals (BL1-N through BL4-N, Appendix Q).

 HUMANS All Human Gene Transfer protocols are currently considered experimental. IBC has not yet established a Human Gene Therapy Advisory Committee to deal with human Gene Transfer studies.

IBC approval must await RAC (NIH Recombinant Advisory Committee) action. Depending on whether the study is deemed “novel” the RAC can either schedule a full examination of the protocol at one of its quarterly meetings or recommend sole FDA review.

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Overcoming the regulatory hurdles involved in gaining approval for a human gene transfer study is not a task for the faint of heart. Beyond approvals from the Food and Drug Administration one has to get approval form the local Institutional Review Board (IRB) and the Biosafety Committee. In addition, RAC will evaluate novel protocols although it does not have approval power. These evaluations often involve the Principal Investigator’s appearance in Bethesda and aggressive questioning by members of the RAC.

For most people this process can be intimidating. Best to get help early through a commercial sponsor or a consultant.

 ANIMALS

Animal Biosafety levels are normally used to cover large animals such as cattle, swine, horses, poultry etc. IBC tends to use the same designations when considering safe practices with lower animals including rodents.

All animal experiments must be reviewed and approved by a local Institutional Animal Care and Use Committee (IACUC). These committees act under the US Department of Agriculture regulations. At Clemson University, the responsible committee is designated the Animal Research Committee (ARC).

 PLANTS

Plant Biosafety levels are necessary when research plants are too big, too many or have growth requirements that cannot be covered by the standard Biosafety Levels. The plant guidelines cover microorganisms and small “animals”, particularly insects, such as arthropods. Plant-associated microorganism include pathogenic viroids, virusoids, viruses, bacteria, fungi, protozoans, as well as benign or beneficial microorganisms known to be associated with plants e.g. (Rhizobium, Pseudomonas).

When studies covered under the plant appendix are being discussed, IBC will include an expert in plant pests or containment.

It is of interest that the plant guidelines are not designed to directly protect humans from plant related recombinant DNA (i.e., the agents covered pose virtually no threat to humans or higher animals). Rather the guidelines are in place to protect the general ecosystem form serious disruption. Thus, procedures are designed to limit the spread of novel organisms form the experimental facility, not to protect the workers.

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SUMMARY OF LABORATORY BIOSAFETY LEVELS

Biosafety Level Risk Group Practices and Techniques Safety Equipment Examples BL1 Individual risk: LOW Standard Microbiological None: primary containment E. Coli K12, Continuous Basic Laboratory Community risk: LOW Practices. provided by adherence to culture of cell lines, e.g., standard lab practices short term, long term during open bench culture of most non- operations. primate mammalian tissue. BL2 Individual risk: Level 1 practices plus: Partial containment (i.e., Hepatitis B Virus, Basic Laboratory MODERATE Lab coats, autoclaving of all Class I or II Biosafety Salmonella typhi, Short With Biosafety cabinets and Community risk: LOW biological waste preferred, cabinets for procedures term, long term culture of other physical containment limited access, biohazard which produce aerosols. human tumor cell lines, devices as required. warning signs on doors and culture of lymphoid lines equipment. carrying inducible EBV, many common human pathogens. BL3 Individual risk: HIGH Level 2 practices plus: Partial containment Yellow fever, M. Containment Laboratory Community risk: Special protective clothing, equipment used for all tuberculosis, Short term with special engineering and MODERATE controlled access through manipulations of infections culture of tissue from non- design features. entrance room, biological materials, directional human primates until waste must be autoclaved, airflow. cultures are known to be preferably in facility. free of Herpes-virus simiae (B. virus) BL4 Individual risk: HIGH Level 3 practices plus: Maximum containment Ebola-Marburg Virus. Maximum Containment Community risk: HIGH Entrance through change equipment (i.e., Class III Propagation of Herpes Laboratory room. Complete change of Biosafety cabinet or partial virus simiae. clothing from street to containment in combination Smallpox. laboratory gear, shower at with full-body, air-supplied exit. All wastes positive-pressure personnel decontaminated on exit suit) used for all procedures from facility. and activities. Clemson University IBC Page 8 of 8

SUMMARY OF ANIMAL FACILITY BIOSAFETY LEVELS

BSL Agents Practices Safety Equipment (Primary Facilities (Secondary Barriers) Barriers) 1 Not known to consistently Standard animal care and As required for normal care of each Standard animal facility. cause disease in healthy management practices, including species. No recirculation of exhaust human adults. appropriate medical surveillance air. Directional air flow programs. recommended. Handwashing sink recommended. 2 Associated with human ABSL-1 practices plus: Limited ABSL-1 equipment plus primary ABSL-1 facility plus: disease. Hazard: access. Biohazard warning signs. barriers: containment equipment Autoclave available. percutaneous exposure, Sharps precautions. Biosafety appropriate for animal species; Handwashing sink available ingestion, mucous membrane manual. Decontamination of all PPES: laboratory coats, gloves, in the animal room. exposure. infectious wastes and of animal face and respiratory protection as Mechanical cage washer cages prior to washing. needed. used. 3 Indigenous or exotic agents ABSL-2 practices plus: Controlled ABSL-2 equipment plus: ABSL-2 facility plus: with potential for aerosol access. Decontamination of Containment equipment for Physical separation from transmission; disease may clothing before laundering. Cages housing animals and cage dumping access corridors. Self- have serious health effects. decontaminated before bedding activities. Class I or II BSCs closing, double-door access. removed. Disinfectant foot bath as available for manipulative Sealed penetrations. Sealed needed. procedures (inoculation, necropsy) windows. Autoclave that may create infectious aerosols. available in facility. PPEs: appropriate respiratory protection 4 Dangerous/exotic agents that ABSL-3 practices plus: Entrance ABSL-3 equipment plus: ABSL-3 facility plus: pose high risk of life through change room where Maximum containment equipment Separate building or isolated threatening disease; aerosol personal clothing is removed and (i.e., Class III BSC or partial zone. Dedicated supply and transmission, or related laboratory clothing is put on; containment equipment in exhaust, vacuum and agents with unknown risk of shower on exiting. All wastes are combination with full body, air- decontamination systems. transmission. decontaminated before removal supplied positive-pressure Other requirements outlined from the facility. personnel suit) used for all in BMBL-4. procedures and activities.